A major factor in diseases caused by Gram-positive (Gr+) pathogens is secretion of toxins and other virulence factors. While the mechanism for transport across the cytoplasmic membrane is known in most Gr+ secretion systems, the process by which secreted proteins traverse the thick Gr+ peptidoglycan (PG) layer is not. Once thought to be produced by only Gram-negative (Gr-) bacteria, our lab discovered that Gr+ bacteria also produce Type IV pili (T4P). Type II secretion (T2S) systems share a high degree of similarity to proteins found in T4P. In Gr- bacteria, the main function of T2S systems is to transport folded proteins from the periplasm through the outer membrane. Clostridium perfringens and many of the Clostridium species examined thus far have two sets of T4P assembly apparatuses. Analysis of one set of T4P-associated genes leads us to hypothesize that they encode the Gr+ equivalent of a T2S system. One protein, CPE0517, which depends on a putative T2S pilin for secretion, binds to host cells, making it a potential virulence factor. The project has two aims. The first aim is to identify and characterize the major components of the T2S system in C. perfringens. In-frame deletions will be introduced into each gene that encodes components of the putative T2S and T4P systems, since they may share some components. The mutants will be tested for loss of secretion of CPE0517, T4P pilus assembly and adherence to host cells. The second aim is to determine the pathway of CPE0517 secretion. Our hypothesis is that CPE0517 undergoes the same overall process for secretion that a T2S substrate would in a Gr- bacteria, but in the context of a Gr+ cell envelope: (1) Sec-dependent translocation across the cytoplasmic membrane (2) folds into its final conformation in the space between the cytoplasmic membrane and PG layer (CM/PG space) and (3) undergoes export across the PG layer via the T2S system. Each of these processes will be tested by (1) Mutating a Sec-dependent signal peptidase that likely processes CPE0517, (2) Determining if CPE0517 forms a complex with other proteins in the CM/PG space and measuring folding of a CPE0517-mCherry protein fusion in the CM/PG space using fluorescence microscopy, (3) using a fluorescence-based kinetic assay to measure the rate of secretion from the CM/PG space. Discovery of a T2S in Gr+ bacteria would be significant, since T2S systems have not been identified in Gr+ bacteria before and would provide a mechanism for export of some proteins through the PG layer. The C. perfringens T2S is also an excellent model to study T2S systems in both Gr+ and Gr- pathogens, since it appears to be very simple, with significantly fewer proteins than those seen in Gr- bacteria.